This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Metalloproteases are a major factor in the toxicity of many pathogenic bacteria including B. anthracis, C. botulinum, and C. tetani. A detailed understanding of the complete kinetic mechanism and factors affecting the site specificity of these proteases will significantly improve our ability to screen and design specific inhibitors of toxic metalloproteases for possible use as therapeutics. Such understanding will also facilitate the creation of sensitive biothreat detection assays that would have a significant impact on homeland defense. This project will develop a flow cytometry based model system that will allow the determination of the detailed kinetic mechanism of a metalloprotease, factors affecting site specificity, and provide a framework for inhibitor screening.